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FOX CHASE CANCER CENTER
OVERALL ABSTRACT
Principal Investigator(s): Robert F. Ozols, M.D., Ph.D.
Co-Investigator(s): Thomas C. Hamilton, PhD
Despite recent treatment improvements, ovarian cancer remains the #1 gynecologic killer in the United States. An expanded translational research effort is necessary to identify new opportunities for diagnosis, prevention, and treatment. The Fox Chase Cancer Center Specialized Program of Research Excellence (SPORE) in Ovarian Cancer consists of seven research projects, a Developmental Research Program, a Career Development Program, and four specialized Cores to support the research programs. The projects are interrelated and are focused on ovarian carcinogenesis and ovarian cancer prevention and treatment: 1) Altered Retinoic Acid Metabolism in Human Ovarian Cancer; 2) Biomarkers of Ovarian Cancer and the Molecular Genetics Analysis of Human Ovarian Surface Epithelial (HOSE) Cell Transformation; 3) The Role of PI 3-Kinase/AKT2 Signaling in Ovarian Oncogenesis; 4) Chemoprevention Studies in Patients at Risk for Ovarian Cancer; 5) Role of DNA-Dependent Protein Kinase in Ovarian Cancer Drug Resistance; 6) Pharmacokinetic-Pharmacodynamic Models to Design and Optimize Clinical Trials in Ovarian Cancer; 7) The Development of an Ovarian-Selective Replicating Adenoviral Vector for use in Gene Therapy for Ovarian Cancer. The last project is in collaboration with the Institute for Human Gene Therapy at the University of Pennsylvania. The specialized Cores include the establishment of an Ovarian Cancer Clinical Network which links FCCC together with the University of Pennsylvania, Hershey Medical Center, Cancer Institute of New Jersey, and other participating institutions to increase accrual to chemoprevention trials for women at high risk for ovarian cancer and to therapeutic trials for women diagnosed with this disease. The Tissue Procurement Core will process biospecimens for molecular studies on oncogenesis and drug resistance. The Genetics Susceptibility Testing Laboratory will screen patients for BRCA1 mutations in order to identify individuals who are eligible for participation in the clinical trial of fenretinide vs. placebo (Project 3). The Administration/Biostatistical Core will provide administrative and biostatistical support for laboratory projects, clinical trials, and for the other Cores. Fox Chase Cancer Center has had a long-term commitment to research in ovarian cancer and will provide additional institutional resources to support the goals of this SPORE application. Senior leadership of the Center is directly involved in the SPORE application. The Principal Investigator is the Senior Vice President for Medical Science. The Co-PI is Program Leader in the CCSG Ovarian Cancer Program and the President of the Center serves on the Executive Committee. This SPORE Program is a multidisciplinary collaboration of laboratory researchers and clinicians focused on decreasing morbidity and mortality from this disease. This goal will be accomplished by prevention strategies based on an understanding of ovarian oncogenesis coupled with novel scientifically-based therapeutic approaches.
PROJECT 1
Altered Retinoic Acid Metabolism in Human Ovarian Cancer
Principal Investigator(s): Thomas C. Hamilton, PhD
Co-Investigator(s): Michael A. Bookman, MD
Our laboratory has had a longstanding interest in unraveling the genetic basis for how ovarian cancer develops and why it progresses. To this end, we have attempted to investigate these issues using approaches that augment work with clinical ovarian cancer specimens. Our strategy has been to develop an in vitro model of ovarian surface epithelial cell transformation based on information about the epidemiology of ovarian cancer. Using this model, we have begun to explore the genetic and gene expression changes that occur between normal rat ovarian surface epithelial cells and their transformed counterparts. In one approach, we have developed and used a modified form of Suppression Subtractive hybridization (SSH) which we refer to as Consolidative SSH. This method is designed to detect gene expression differences consistently present between four independently transformed rat ovarian surface epithelial (ROSE) cell lines and their normal counterpart. Of direct potential relevance to our SPORE in Ovarian Cancer was the outcome of investigation of genes whose expression was consistently lost in the four transformed ROSE cell lines. This study revealed that the expression of two genes homologous to human genes important in retinol metabolism, retinol binding protein (RBP, GenBank accession # X00129 ) and cellular retinol binding protein I (CRBPI, GenBank accession # M11433) was consistently lost in the transformed ROSE cells. It has been suggested that CRBPI plays a critical role in the intracellular conversion of vitamin A/retinol to retinoic acid. Based on the established role of retinoids as physiological molecules that participate in regulation of the redox status of the intracellular space and inhibition of transformation, we hypothesize that the loss of RBP and/or CRBPI in our ROSE ovarian cancer model results in the decreased functionality of a physiologically significant mechanism that inhibits carcinogenesis. This is consistent with a clinical trial of fenretinide which showed a decrease in ovarian cancer incidence in women treated with the drug compared to placebo. Additionally, one of our projects is exploring the potential of fenretinide to reverse ovarian preneoplasia in cancer-prone ovaries. Hence, it seems especially timely to explore whether altered retinol metabolism is a pathway involved in malignant transformation of the ovarian surface epithelium.
PROJECT 2
Biomarkers of Ovarian Cancer and the Molecular Genetic Analysis of HOSE Cell Transformation
Principal Investigator(s): Andrew K. Godwin, PhD
Co-Investigator(s): Mary B. Daly, MD, PhD
The goal of this project is to elucidate the genetic and biological determinants of ovarian cancer, focusing on an in vitro model for ovarian cancer that we have developed (1-4). First, we propose to identify genes that are differentially expressed upon malignant transformation of HOSE cells using a modified suppression subtractive hybridization (SSH) approach. We will use consolidate SSH (CSSH) to identify genes that are either overexpressed or underrepresented upon immortalization and malignant transformation of HOSE cells. Second, we propose to identify genes that are differentially expressed in HOSE cells heterozygous for BRCA1 (+/-) using CSSH. We have previously reported that the surface epithelial cells from ovaries of BRCA1 mutant allele carriers have a different phenotype in vitro compared to control surface epithelial cells and these features are present without the inactivation of the remaining wild-type allele of BRCA1. These results suggest that other genetic changes might precede loss of both copies of BRCA1 in the genesis of familial ovarian cancer. We will compare populations of HOSE cells for differentially expressed gene related to their BRCA1 carrier status. Third, we propose to determine the expression pattern of candidate ovarian cancer-causing genes identified by CSSH in benign and borderline ovarian tumors and ovarian carcinomas. To find the subset of genes discovered in our tissue culture model which are relevant to clinical ovarian tumors, cDNA arrays representing the differentially expressed clones will be created. Reverse transcribed RNAs derived from benign, borderline, early and late stage ovarian tumors will be hybridized to individual arrays and the patterns of expression determined. Clones showing expression patterns consistent with the findings in the model will be evaluated for tissue specific expression by multiple tissue northern blot analysis. Fourth, we propose to establish the pattern of expression of candidate ovarian cancer-causing genes in preneoplastic lesions by performing in situ hybridization on cancer prone ovaries. We have previously identified a preneoplastic phenotype in the ovaries from women at increased risk for developing ovarian cancer. Once we have confirmed the expression of several genes, in situ hybridization and immunohistochemical approaches will be used to evaluate their RNA and protein expression patterns at potentially the earliest stages of cancer development. Overall, the studies proposed should enable us to 1) determine if the histological phenotype we have previously described is likely to be a true precursor of ovarian cancer; 2) establish whether other genetic changes precede BRCA1 inactivation in ovarian carcinogenesis; 3) identify genes that are specifically expressed in the ovarian surface epithelium as opposed to the entire ovary; and 4) identify early surrogate intermediate endpoint biomarkers of impending ovarian cancer.
PROJECT 3
The Role of PI3-kinase/AKT2 Signaling in Ovarian Oncogenesis
Principal Investigator(s): Joseph R. Testa, PhD
Co-Investigator(s): Matthew Boente, MD
AKT2 is a member of the Akt/protein kinase B family and is activated in response to phosphatidylinositol 3-kinase (PI3-K)-mediated signals triggered at the cell membrane by growth factors. Amplification and/or overexpression of the AKT2 oncogene is associated with the development of human ovarian carcinomas. Therefore, understanding the role of AKT2 and PI3-K-mediated signals in the growth regulation of human ovarian surface epithelial (HOSE) cells will contribute to a better understanding of the molecular mechanisms underlying ovarian oncogenesis, which is the broad, long-term objective of this project. The specific aims are: 1) Determine the involvement of AKT2, the related AKT1, and PI3-K in human ovarian cancer. Examine the expression/kinase activity of AKT2, AKT1, and the catalytic subunit of PI3-K (p110a) in ovarian carcinoma specimens, and their expression in premalignant lesions in women predisposed to ovarian cancer. Correlate these laboratory findings with various clinicopathologic parameters to determine their clinical relevance. 2) Characterize the role of AKT2 in the regulation of physiological and neoplastic growth of HOSE cells in vitro. Evaluate the effect of the expression and/or activity of AKT2 on growth rate, cell cycle progression, and the induction of apoptosis. Using cDNA expression arrays, identify groups of genes associated with ovarian oncogenesis, whose expression is regulated in response to AKT2 expression/activity. 3) Determine the role of AKT2 in invasion and metastasis of HOSE cells and ovarian cancer cells. Using conditional retroviral expression constructs, investigate the potential involvement of AKT2 expression/activity in cell survival and the development of more invasive or metastatic phenotypes. 4) Identify modulators and substrates of AKT2. Using the yeast two-hybrid system and ovarian cancer-specific phage cDNA expression libraries, identify proteins that interact with AKT2 in the transduction of mitogenic signals in ovarian cancer cells, and determine the physiological significance of these interactions in HOSE cells. Overall, the studies proposed here will provide important new insights regarding the mechanisms by which AKT2 and PI3-K-mediated signals contribute to ovarian oncogenesis, and may create potential avenues for therapeutic intervention.
PROJECT 4
Implementation of cDNA Array Techniques and Pattern Recognition Analysis to Study Ovarian Cancer Development and Chemoprevention
Principal Investigator(s): Christos Patriotis, PhD
Co-Investigator(s): Paul F. Engstrom, MD
Growing out of the human genome project, the cDNA expression arrays are a very powerful genetic technique for the identification and the differential display of genes expressed in different cells. This young technology, still undergoing rapid development, allows the assessment of the expression of hundreds to thousands of genes simultaneously. An impediment of this technique is that it generates large amounts of data, which at the present state of software for analysis, are difficult to interpret. The development of adequate algorithms for processing the data in its full complexity should significantly increase our ability to maximally extract information from cDNA array data sets. This perhaps will allow the prediction of statistically probable gene expression patterns, characteristic of cells traversing the continuum between normal and malignant or responding to specific growth conditions. The goal of this Project is to introduce the array technology into the Ovarian Cancer Research Program at Fox Chase Cancer Center and, by drawing from our Institute's extensive experience, to implement highly sensitive pattern recognition algorithms to cDNA array-obtained information. The proposed study will position this important new technology within our Ovarian Cancer Research Program so that it is readily available to address many other important questions more easily and thoroughly than is currently possible. To meet the specific goals of this study the following Specific Aims will be pursued: 1) Determine and further optimize the conditions for reproducibility of the hybridization patterns obtained on commercial arrays using replicate cDNA probes synthesized with identical mRNA preparations from HOSE cells; 2) Implement pattern recognition algorithms which will differentiate between the mRNA expression patterns of normal HOSE cells and the same HOSE cell lines experimentally induced to undergo malignant transformation; 3) Determine the pattern of gene expression in HOSE cells treated in vitro with Fenretinide (4-HPR). By implementing these specific aims, the present study will obtain important information with regard to the patterns of gene expression and how they change as ovarian cancer develops and is treated with chemopreventive agents.
PROJECT 5
Role of DNA-Dependent Protein Kinase in Ovarian Cancer Drug Resistance
Principal Investigator(s): Kenneth D. Tew, PhD, DSc
Co-Investigator(s): Robert F. Ozols, MD, PhD
Chemotherapeutic treatment of ovarian cancer remains an integral component in disease management. Many of the protocols used include combinations of platinum-based drugs together with paclitaxel. More recent trials suggest that anthracyclines also have significant activity in combination therapy. This component of the SPORE grant is designed to address how resistance to these agents may occur and limit successful therapy. We have secured preliminary data to suggest that platinum- and adriamycin-resistant cells have enhanced expression of DNA-dependent protein kinase (DNA-PK), an enzyme complex with bi-functionality in cell signaling and DNA repair. These properties make the enzyme a particularly attractive subject for study in ovarian cancer models of acquired drug resistance. Our aims will include: understanding the expression of the catalytic subunit (DNA-PKcs) and the Ku autoantigen (Ku70 and Ku80) in ovarian cancer cell lines acutely and chronically exposed to cisplatin or adriamycin; extend these in vitro studies into archival and fresh human ovarian biopsy tissues; define the transcriptional and/or post-transcriptional mechanism(s) by which cells increase expression of DNA-PK; compare and contrast the DNA repair properties of the enzyme with the kinase signaling properties; the latter will be determined, in part, by the identification of DNA-PK mediated phosphorylated downstream proteins; characterization of the nature of the DNA-PK inhibitory properties of novel glutathione peptidomimetic drugs; these agents will then be tested as modulators of resistance and preclinical data will be gathered with a view to clinical trial design and implementation. Successful completion of these goals should provide for rational means of improving and extending existing drug treatment approaches for ovarian cancer.
PROJECT 6
Pharmacokinetic-Pharmacodynamic Models to Design and Optimize Clinical Trials in Ovarian Cancer
Principal Investigator(s): Michael A. Bookman, MD
Co-Investigator(s): James M. Gallo, PhD
Chemotherapy of women with advanced-stage ovarian cancer achieves good initial results with only a limited impact on long-term disease outcomes, as over 80% of these patients eventually develop recurrent disease accompanied by a progressive increase in drug-resistant tumors. Long-term outcomes might be improved through the development of new chemotherapy regimens, especially through incorporation of agents that exhibit pharmacological synergy. Toxicity of these new regimens has been substantial, and management of hematologic toxicity has thus assumed the dominant role in clinical trial design, without direct or indirect evidence that the desired biologic targets are actually being modulated within the tumor. The number of potential combinations and sequences is daunting, and many promising regimens will be overlooked using a conventional empiric approach to clinical trial design. The overall goals of this project will be to apply and integrate pharmacokinetic (PK) and pharmacodynamic (PD) principles in both preclinical models and clinical trials to select and optimize combination chemotherapy in ovarian cancer patients. We proposed to utilize a syngeneic rat model of advanced epithelial ovarian cancer as a translational tool to address biologic questions related to new combination chemotherapy regimens. Hybrid physiologically based (PB)-PK/PD models will be developed to optimize the therapeutic index of combination regimens in the rat model. These animal-derived models will then be extrapolated to human ovarian cancer patients through focusing on key target organs such as tumor and bone marrow. In this manner, the human PB-PK/PD models can rationally select promising combination protocols that will undergo phase I clinical evaluation in newly diagnosed patients using local and national resources in collaboration with the Gynecologic Oncology Group. PK/PD measurements in patients will be integrated in a population-based PK-PD modeling strategy to identify and account for patient variables. This will further refine the treatment regimen and facilitate the design of individualized drug regimens. Overall, it is proposed that through the quantitative nature of PK/PD models, identification of rational combination regimens with significant impact on ovarian cancer will be expediated.
PROJECT 7
Development of an Ovarian-Selective Replicating Adenoviral Vector for Use in Gene Therapy for Ovarian Cancer
Principal Investigator(s): Steven M. Albelda, MD
Co-Investigator(s): Stephen C. Rubin, MD
New treatments for ovarian cancer are clearly needed. One novel approach under active preclinical and clinical evaluation is gene therapy. Strategies being investigated include use of replication incompetent retroviruses or adenoviruses (AD) to deliver suicide genes such as herpes simplex virus (HSV) thymidine kinase (tk) to activate ganciclovir (GCV) into a cytotoxic drug. One major limitation discovered in an ongoing Phase 1 trial for malignant mesothelioma at the University of Pennsylvania is poor depth of penetration of ADHSVtk into the tumor after intracavitary delivery. A promising approach to overcome this problem is to use replication-competent adenoviruses. When such viruses infect cells and replicate, it causes cell lysis. In addition, active virus is released to infect other tumor cells. By coupling this mechanism of enhanced killing and infection with the ability to activate GCV, we hypothesize that anti-tumor efficacy will be enhanced. However, the delivery of such replication competent viruses causes some safety concerns. To address this issue, a virus conditionally replicative in tumor cells will be constructed. Success would result in a clinical gene therapy trial. Based on the recent discovery at Fox Chase Cancer Center of a promoter which shows specificity of function in human ovarian cancer, the goal of this proposal is to construct such vectors and preclinically evaluate their efficacy and safety by accomplishing the following specific aims: Specific Aim 1. Develop and evaluate a replication-competent adenoviral vector expressing HSVtk. This will be accomplished by developing and testing a replicating adenoviral vector containing the HSVtk suicide gene. In a first series of experiments (proof of principal), we will study a fully replicative virus containing the HSVtk gene inserted into the E3 region in ovarian tumor models. This will begin to allow us to understand the dynamics of viral replication vs delivery of GCV. Specific Aim 2. Develop and evaluate a replication-competent adenoviral vector expressing HSVtk that will only replicate in ovarian cancer cells. This will be accomplished by developing Ad mutants that replicate selectively in ovarian tumors using the "U3" promoter developed by Dr. Hamilton and his group. These vectors will be made by disrupting the normal Ad E1a promoter region and inserting the ovarian cancer-selective promoter into this region. Since replication is dependent on early production of E proteins, viral replication will be limited to those cells in which the tumor specific promoter is active.
CORE 1
Ovarian Cancer Clinical Network
Principal Investigator(s): Mary B. Daly, MD, PhD
Co-Investigator(s): Eric A. Ross, PhD
The potential to understand ovarian cancer at the most basic molecular level poses a challenge to the scientific community to apply the new and developing technologies to advance our understanding of this deadly disease. What has been lacking is the opportunity to prospectively follow a sufficiently large cohort of women with ovarian cancer or with a familial risk for ovarian cancer to definitively address these questions. The Fox Chase Cancer Center (FCCC) proposes the establishment of The Ovarian Cancer Clinical Network Core, a research-based infrastructure to facilitate the conduct of translational research and to transfer novel prevention, screening and treatment strategies into clinical practice. This core represents a partnership of FCCC, the Fox Chase Network, Cooper Medical Center, Temple University, the Cancer Institute of New Jersey, Hershey Medical Center, and the University of Pennsylvania, and provides access to a wide spectrum of ethnic, socioeconomic and rural/urban populations. Building upon an established infrastructure of over 1,600 high-risk breast cancer families and using the combined resources of the Core facility, a cohort of families with histopathologic documentation of one or more cases of ovarian cancer will be assembled. Extensive information on medical, reproductive, family and lifestyle factors will be collected. Blood samples will be collected from all participants. Ovarian tissue specimens will be requested from affected individuals as well as those unaffected family members undergoing oophorectomy for prophylaxis. The Core will design, develop, and support a comprehensive information management system to ensure accurate data entry and maintenance and to serve the needs of the SPORE investigators. Counseling and screening protocols will be standardized and disseminated to health care professionals. This facility will build upon the seminal work underway at Fox Chase to serve the needs of high-risk families and will expand these efforts to foster multidisciplinary research projects. The creation of a dynamic ovarian cancer research database provides a unique opportunity to address multidisciplinary research questions and to bring the knowledge about ovarian cancer generated by the genetic revolution to the community.
CORE 2
Tissue Procurement
Principal Investigator(s): Andres Klein-Szanto, MD
This Core is intended to be a resource for the SPORE as a whole and a valuable source of biological materials for the present and future projects and pilot studies. The laboratory core will be responsible for the collection, storage, and distribution of ovarian tissues and tumors. Fresh/frozen samples will be collected at Fox Chase Cancer Center and other participating sites and forwarded to our Core laboratory. This laboratory Core will also be responsible for the collection of archival ovarian tumor specimens and the extraction of DNA to be distributed to the participating projects and pilot studies. It is estimated that more than 400 new ovarian cancer cases will be identified each year through the Ovarian Cancer Clinical Network (Core 1) during the course of our proposal. It is very conservatively estimated that approximately 30% of these cases will be accrued as fresh/frozen specimens. Paraffin-embedded tumors will be available from all cases. Tumor samples will be used by Projects 1, 2, 3 and 5. In addition, we will collect ovaries removed for prophylaxis from women considered to be at increased risk of developing ovarian cancer (e.g., BRCA1, BRCA2 mutant allele carriers, or suggestive family history of cancer) who participate in our chemoprevention trial. These tissues will be processed and distributed for pathology and immunohistochemical evaluation to determine the effect of fenretinide and other chemopreventive agents on the cancer-prone phenotype that we have previously observed in at-risk ovaries. By centralizing these services into a Core, we will be better able to manage and coordinate the collection, storage, and distribution of a large number of highly valuable tumor, cancer-prone, and normal specimens.
CORE 3
Genetic Susceptibility Testing Laboratory
Principal Investigator(s): Andrew K. Godwin, PhD
This Core is intended to be a resource for the SPORE as a whole and a valuable source of biological materials and genetic information for our continuing studies of the etiology of ovarian cancer. The Genetic Susceptibility Testing Laboratory Core will be responsible for the processing of blood samples collected through the "Ovarian Cancer Clinical Network" Core directed by Dr. M. Daly. Biological samples collected at all participating sites will be forwarded to the Clinical Molecular Genetics Laboratory at Fox Chase Cancer Center for processing and banking under CLIA approved guidelines. It is anticipated that approximately 4,300 peripheral blood samples will be collected and processed during the course of the proposed studies. These samples will come from women diagnosed with ovarian cancer and their first- and second-degree female relatives. A subset of these samples will be from breast cancer and breast/ovarian cancer syndrome families and will be tested for germline mutations in BRCA1. The Genetic Susceptibility Testing Laboratory Core will also be responsible for the distribution of constitutional DNA samples from ovarian cancer patients to SPORE participants. Ovarian tumor DNA and corresponding normal (from peripheral blood) will be evaluated by Dr. Hamilton for loss of heterozygosity on chromosome 6q and for mutational analysis of LOT1. Blood samples will also be used in several pilot studies. Dr. Godwin will use these samples to determine if the pattern of gene expression differs between sporadic and hereditary forms of ovarian cancer. Overall, this Core is a natural extension of our long-term interest in the etiology of ovarian cancer, and our Clinical Molecular Genetics laboratory will serve as the Genetic Susceptibility Testing Laboratory Core for the collection, processing, storage, and distribution of a large number of highly valuable biospecimens, and for the BRCA1 mutation testing of women participating in chemoprevention trials.
CORE 4
Administration/Biostatistics
Principal Investigator(s): Robert F. Ozols, MD, PhD
Co-Investigator(s): Thomas C. Hamilton, PhD
The Administration/Biostatistics Core of the FCCC Ovarian SPORE will be responsible for oversight of all administrative, financial, and scientific activities of the SPORE Program. The Core will also provide biostatistical support for all Projects, Pilot Projects and other Cores. The Core will monitor and regulate the financial expenditures and prepare detailed financial records. The Core will also be responsible for planning and evaluation as well as promoting scientific interchange among members of the SPORE including: 1) scheduling the monthly scientific meeting of project investigators; 2) coordinating the scientific retreat; 3) assisting in travel arrangements for External Advisory Committee; and 4) providing travel funds for SPORE leaders to attend the Annual SPORE Meeting as well as for Dr. Ozols to attend the annual SPORE Directors' Meeting at the NCI. The Core will provide secretarial support to the SPORE Executive Committee, Developmental Research Committee and Career Development Committee. The Core will also maintain a record of all publications and presentations derived from the SPORE investigators relating to ovarian cancer. The Administration/Biostatistics Core will also be responsible for ensuring the optimum utilization of SPORE resources to prevent duplication with existing P30 CCSG Cores and Facilities.